Hiv regulatory and auxiliary peptides, antigens, vaccine compositions, immunoassay kit and a method of detecting antibodies induced by hiv

ABSTRACT

The present invention comprises novel and modified peptides capable of inducing a HIV-1 specific immune response without antaonizing the cytotoxic T-cell activity in order to achieve an effective prophylactic and therapeutic vaccine against HIV. The peptides are based on conserved regions of HIV Tat and Rev, regulatory proteins and Nef, auxiliary proteins. Antigens in free- or carrier-bound form comprising at least one of the said peptides, vaccine compositions containing at least one of the antigens, immunoassay kits and a method of detecting antibodies induced by HIV or HIV specific peptides using such antigens, are described.

The present invention relates to novel peptides based on conservedregions of the regulatory and auxiliary HIV proteins, antigens in freeor carrier-bound form comprising at least one of the said peptides,vaccine compositions containing at least one of the antigens,immunoassay kits and a method of detecting antibodies, induced by humanimmunodeficiency virus (HIV) or HIV-specific peptides, using suchantigens.

BACKGROUND

Human immunodeficiency virus type 1 (HIV-1), the causative agent ofacquired immunodeficiency syndrome (AIDS) continues to present aformidable challenge to health in developing countries. In the Westernworld, therapeutic strategies that target HIV-1 replication andmaturation have had a prominent impact on disease progression. The highcost of current treatment, high toxicity of the drugs and lack of cure,however, means that the development of safe and effective vaccinesremains paramount for control of the AIDS pandemic.

HIV-1 is a complex retrovirus encoding six regulatory and auxiliarygenes not found in the simple retroviruses, namely, tat, rev, nef, vif,vpr and vpu (Table 1). In eukaryotic cells, only completely splicedmRNAs are exported to the cytoplasm for translation. Unspliced orpartially spliced RNAs are retained and eventually degraded in thenucleus. In this way, proteins encoded by the tat, rev and nef genes(designated Tat, Rev and Nef) derived from multiply spliced RNA species,are expressed first and constitute early HIV-1 gene expression. In orderto express singly spliced RNAs and also to transport the full lengthunspliced RNA genome into the cytoplasm for packaging, HIV-1 hasdeveloped means to overcome the restrictions on RNA transport. Theregulatory proteins, Tat and Rev, are essential for HIV-1 replicationsince mutations in these proteins eliminate HIV-1 production (Dayton A.I., et.al. (1986) Cell, 44:941-947, and Fisher, A. G., et.al. (1986)Nature, 320:367-371.).

The auxiliary genes are derived from exons positioned entirely upstreamof the HIV-1 envelope gene e.g. vif or exons upstream of as well aswithin env but in different reading frames e.g. tat, rev. The efficiencyof splicing in part regulates the levels of gene expression of thedifferent auxiliary proteins.

Following integration of HIV-1 proviral DNA, predominantly truncatedforms of mRNA are synthesised by the cellular RNA polymerase II whichinteracts with sites on the 5′ long terminal repeat (LTR) of theproviral DNA. tat is one of the first genes to be expressed and carriesa nuclear localisation signal. It is a potent transcriptional activatorthat enhances LTR-directed transcription up to a thousand fold.Continuous expression of Tat ensures a positive feed back loop forcontinued high level gene expression. Unlike conventionaltranscriptional activators that interact with DNA sequences, Tat bindsdirectly to the 5′ ends of all HIV-1 RNAs at a specific stem-loopsecondary structure, TAR (transactivating response element). Thestructure of the loop is highly conserved and essential for Tatfunction. The structure of the Tat/TAR interaction has been analysedusing nuclear magnetic resonance (NMR) (Puglisi et al. (1992) Science,257:76-80). Tat binds TAR in association with the cellular protein,cyclin T, which in turn binds CDK9 that phosphorylates the RNApolymerase II C-terminal domain, thereby promoting the elongation of RNAtranscripts. These Tat cellular cofactors are only present in activatedcells, their absence represses transcription of proviral DNA resultingin a ‘quasi latency’ in T lymphocytes. Tat is expressed from two exons,both the nuclear localisation signal and the TAR binding region arelocated in the first exon. Tat is secreted from infected cells and canexert heterologous effects on neighbouring cells. These include cellularactivation (Hofman et al. (1993) Blood,. 82:2774-2780.), induction ofcellular apoptosis (Macho et al. (1999) Oncogene, 18:7543-7551.1999)functioning as a secretable growth factor (Trinh, D. P. et.al. (1999)Biochem. Biophys. Res. Commun., 256:299-306.) and modulating host cellprotein synthesis in favour of viral protein synthesis (Xiao et al.(1998) Biochem. Biophys. Res. Commun., 244:384-389.1998). Therapeuticstrategies targeting Tat will therefore have a strong impact on HIV-1infection.

The small multiply spliced mRNAs encoding Tat, Rev and Nef predominateduring early phase after infection. When a threshold level of Rev isproduced, unspliced and singly spliced RNAs accumulate in the cytoplasmfor translation, allowing productive infection to proceed. Failure togenerate this threshold level of Rev may contribute to HIV quasilatency. Rev can only bind to RNAs carrying an RNA structure, RRE (Revresponsive element) which is located in the env coding region of thegenome. Rev interacts with the RRE as a multimer through a basicarginine rich region present in the amino terminal half of the protein.A consequence of this interaction is the transport of partially splicedRNAs that will provide gene products such as Env, Vif, Vpu and Vpr aswell as unspliced RNAs that will serve as new genomes for incorporationinto assembling particles. Structural analysis of the Rev/RREinteraction has also been carried out using NMR (Battiste et al. (1996)Science, 273:1547-1551.1996). Rev carries a leucine rich export signalthat allows it to shuttle between the nucleus and the cytoplasm forcontinued transport of newly synthesised RNAs (Kalland et al. (1994). J.Virol., 68:1475-1485; Meyer & Malim, (1994) Genes Dev., 8:1538-1547.).In this way, Rev ensures that the structural genes are expressed latefollowing the regulatory genes. Therapeutic strategies directed againstRev will interrupt the viral life cycle early in infection.

Although originally described as a negative factor, Nef has later beenshown to have positive effects on virus replication and is expressed inlarger quantities than that of Tat and Rev, both early and throughoutinfection. Nef is myristylated at the N-terminus and is associated withthe inner side of the plasma membrane. Nef is partly responsible fordown regulation and degradation of surface CD4 by endocytosis (Piguet etal. (1998) EMBO J., 17:2472-2481.). Removing CD4 from the cell surfaceprevents superinfection with other HIV-1 strains, or reinfection withnewly released virus. Nef is also responsible for the down regulation ofMHC class I thereby protecting infected cells from destruction bycytotoxic T-lymphocytes (Le Gall et al. (1997). Res. Virol.,148:43-47.). Nef is not an essential viral protein since it is notrequired for in vitro infection of peripheral blood lymphocytes orT-cell lines. Nef deletion mutants, however, are less pathogenic overlong periods of time. Nef also has complex effects on signaltransduction pathways in the cell and contains a proline rich regionthat can interact with the SH3 domain of kinases involved in T-cellactivation, a feature necessary for efficient HIV replication (Moarefiet al. (1997). Nature, 385:650-653.). Nef containing viruses are capableof more viral DNA synthesis than viruses deleted in the Nef gene whichsuggests that Nef directly or indirectly activates the viral reversetranscriptase. The low level of Nef associated with virions may beresponsible for this phenomenon. Low levels of Nef are also releasedfrom infected cells although the potential effect on neighbouring cellsis unclear. Since Nef is expressed early in infection and hassignificant effects on CD4 and MHC class I expression as well as diseaseprogression, it represents an important target for future therapeuticstrategies.

Tat and Nef are secreted and can be taken up by macrophages andexpressed in association with MHC class II molecules. This improvestheir suitability as targets for peptide based therapies which alsowould be expressed in the context of MHC class II. It is clear thattargeting early gene products that are essential for HIV-1 replication,such as Tat and Rev should be given priority in addition to Nef which isalso expressed early and influences disease progression. TABLE 1 HIV-1Regulatory and auxiliary proteins. Gene Protein Name ExpressionLocalisation Functions Tat Tat Transactivator Early Nucleus Activatesviral of viral transcription. transcription Secreted from infected cellswhere it can activate T-cells, induce apoptosis and function as a growthfactor. Rev Rev Nuclear RNA Early Nucleolus, Regulates export factornucleoplasm, splicing/RNA cytoplasm transport to the cytoplasm. Ashuttle protein. Nef Nef Numerous Early Cytoplasm, Triggers CD4 effectormembrane endocytosis, Down functions associated. regulates MHC classVirions 1 expression. Binds to kinases and may influence T-cellsignalling and activation. Vpu Vpu Viral protein u Late Cytoplasm,Triggers intracellular membrane CD4 degradation, associated downregulates MHC class 1, nonspecific promoter of retroviral particlerelease. Vif Vif Viral infectivity Late Cytoplasm, Enhances infectivityfactor membranes of viral particles in a Virions cell dependent manner.Improves viral DNA synthesis during reverse transcription. Vpr Vpr Viralprotein r Late Predominantly Contributes to nucleus nuclear import ofVirions preintegration complex. Arrests cells in G2/M phase of the cellcycle.

Naturally occurring HIV sequences in vaccine candidates are not capableof stimulating a stable longterm immune response due to HIVs inherentability to hide by changing the appearance of the epitopes presented forthe immune system. To overcome this variable presentation of epitopes,certain amino acid substitutions and amino acid combinations willsupport the immune system to present and recognize these foreign virusantigens in a reliable manner and thus to a greater extent.

Based on the above background, we decided to investigate the possibilityof designing novel synthetic peptides which can mimic the epitopes fromthe regulatory and auxiliary HIV proteins in such a way that they can beexposed for both the humoral as well as the cellular part of the immunesystem, to meet the need for an effective therapeutic and/orprophylactic vaccine.

The initital work was based on the native Tat amino acid sequencespublished by Korber B., et al., Human Retroviruses and AIDS 1997Eds.Theoretical Biology and Biophysics Group, Los Alamos NationalLaboratory, Los Alamos, N. Mex. The first Tat epitope is located betweenamino acid 1 and amino acid 24 of the tat protein: TABLE 2 Tat epitopeAA no Naturally occurring AAs 1 M S 2 E D V 3 S Q P L V A 4 V I 5 D N 6P H A 7 R N S K E D 8 L I Q R V M T 9 E D P 10 P S 11 W 12 K N E H L 13H R Q 14 P 15 G P 16 S N A 17 Q K T 18 P H 19 K T S A R P E Q 20 T A I21 A P D V 22 C S 23 T N S 24 N K R Q A P T

The one letter as well as the three letter codes defining the aminoacids in the sequences given throughout this specification are inaccordance with International standards and given in textbooks, forinstance Lehninger A. L., <<Principles of Biochemistry>>, WorthPublishers Inc., New York, 1982. The amino acids given to the right ofthe left column, represent the natural variation of the sequence. Ouranalyses resulted in a sequence containing this modified epitope: C S WV N P R L E P W L H P G S Q P NI T A C T N|__________________________________________|

Wherein NI indicates 2-aminohexanoic acid (Norleucine, abbreviated Nleand NI in the three letter and one letter code, respectively) and thecysteine residues are in an oxidized state, i.e. are forming anintrachain disulphide bridge. Since the Cystein residue to theC-terminal part (in position 22) of the peptide is part of anintramolecular disulfide bond outside this selected epitope, a similarintrapeptide disulphide bond is formed by placing a Cystein in theN-terminal part of the selected epitope. Another alternative is to forman intermolecular disulphide bond by dimerization of the sequences: W VN P R L E P W L H P G S Q P NI T A C T N                                       | W V N P R L E P W L H P G S Q PNI T A C T N

A further alternative is dimerization with another epitope selected fromTat. The second epitope on Tat is located between amino acid 35 and 57in C-terminal direction, separated from the first epitope by 10 aminoacids containing 5 Cys residues in addition to the Cys residue in eachof the epitopes. The relatively high number of Cys residues offers avariety of inter- and intramolecular crosslinking possibilities. It islikely that this Cys-rich domain will dominate the immunologicalexposure of this protein and hence cause a “hiding” of the two relevantepitopes. Selection and modification of the two adjacent epitopes canexpose an essential part of the Tat protein in a more optimal way.

In order to reduce the probability for development of escape mutants,the number of epitopes is further increased and two additional peptidesequences were selected. These sequences are located on Rev (residues58-78) and Nef (residues 65-85). The native sequences have beenpublished in Human Retroviruses and AIDS 1999; A Compilation andAnalysis of Nucleic Acid and Amino Acid Sequences. Eds.TheoreticalBiology and Biophysics Group, Los Alamos National Laboratory, LosAlamos: TABLE 3 Tat epitope AA no. Naturally occurring AAs 35 Q P I L TY V 36 V A C L 37 C 38 F 39 I L Q M T 40 T N K R 41 K Q 42 G A 43 L 44 GS 45 I 46 S F Y 47 Y N 48 G 49 R K S 50 K 51 K 52 R 53 R K S G 54 Q R P55 R 56 R 57 R G S

TABLE 4 Rev epitope: AA no. Naturally occurring AAs 58 R W Q 59 I V L F60 L I P 61 G S N D C V T A R 62 T A D N S 63 Y C F R H S V L 64 L V 65G H 66 R G 67 S P F L 68 A T E Q V P S 69 E K Q D N 70 P S A N 71 V N GP 72 P Q H S L R T D I 73 L F V 74 Q L P H D E 75 L 76 P 77 P L E 78 L IV P

and the TABLE 5 Nef epitope, Table 5: AA no. Naturally occurring AAs 65E G D S 66 N G D E 67 E V 68 A G 69 L F 70 P 71 I V 72 T R K A M 73 P 74Q H 75 V L I 76 P 77 L V T 78 R 79 P 80 M V I 81 T D 82 Y F R 83 K R 84A G S E Q 85 A S V

Several modified peptides have been synthesized in order to determinethe uniqueness of the sequences as well as their properties forstimulation of the immune system in combination with their specificityand sensitivity as HIV-1 antigens.

DESCRIPTION OF THE INVENTION

The peptides according to the invention are originating from the fourdifferent conserved areas of the HIV-1 Tat, Rev and Nef proteins whichare described above, having the properties of maintaining the uniqueness(immunogenicity, sensitivity and specificity) of the HIV-1 epitopes.Further the new peptides according to the invention possess norecognized cytotoxic T lymphocyte (CTL) antagonistic effect and shallhave at least one potential CTL epitope.

The peptides, according to the invention, which have met the abovecriteria are from the following groups; Xaa₁ Xaa₂ Xaa₃ Xaa₄Xaa₅ Xaa₆Xaa₇Leu (SEQ ID NO: 1) Glu Pro Trp Xaa₁₂ His Pro Xaa₁₅ Xaa₁₆ Xaa₁₇ Xaa₁₈Xaa₁₉ Xaa₂₀ Xaa₂₁ Xaa₂₂ Xaa₂₃ Xaa₂₄wherein the amino acids of the chain could have the following meanings;

-   -   Xaa in position 1 of the peptide derivative is Met, Ser, Cys or        none,    -   Xaa in position 2 is Glu, Asp, Val, Ser or none    -   Xaa in position 3 is Ser, Gln, Pro, Leu, Val, Ala, Trp, Tyr or        Phe,    -   Xaa in position 4 is Val or Ile,    -   Xaa in position 5 is Asp, Asn or Ile,    -   Xaa in position 6 is Pro, His or Ala,    -   Xaa in position 7 is Arg, Asn, Ser, Lys, Glu or Asp    -   Xaa in position 12 is Leu, Ile or Nle    -   Xaa in position 15 is Gly or Pro    -   Xaa in position 16 is Ser, Asn or Ala,    -   Xaa in position 17 is Gln, Lys or Thr    -   Xaa in position 18 is Pro or His,    -   Xaa in Position 19 is Lys, Thr, Ser, Ala, Arg, Pro, Glu, Leu,        Ile or Nle    -   Xaa in position 20 is Thr, Ala or Ile    -   Xaa in position 21 is Ala, Pro, Asp or Val    -   Xaa in position 22 is Cys or Ser    -   Xaa in position 23 is Thr, Asn or Ser    -   Xaa in position 24 is Asn, Lys, Arg, Gln, Ala Pro or Thr        the peptide comprises at least six consecutive amino acids of        the sequence of SEQ ID NO: 1,

furthermore two or more of the Cys residues may form part of anintrachain- or interchain disulphide binding, a —S—(CH₂)_(p)—S— or a—(CH₂)_(p)-bridge wherein p=1-8, optionally intervened by one or morehetero atoms such as O, N or S, Xaa₁ Xaa₂ Xaa₃ Phe Xaa₅ Xaa₆ Xaa₇ (SEQID NO: 4) Xaa₈ Xaa₉ Xaa₁₀ Xaa₁₁ Xaa₁₂ -Z- Tyr Xaa; Gly Xaa₁₅ Lys Lys ArgXaa₁₉ Xaa₂₀ Xaa₂₁ Xaa₂₂ Xaa₂₃wherein the amino acids of the chain have the following meaning;

-   -   Xaa in position 1 is Pro, Ile, Leu, Thr, Tyr or Val    -   Xaa in position 2 is Val, Ala Cys, Leu,    -   Xaa in position 3 is Cys, Ile, Leu, Val or Nle    -   Xaa in position 5 is Ile, Leu, Gln, Met or Thr    -   Xaa in position 6 is Thr, Asn, Lys or Ara    -   Xaa in position 7 is Lys, Arg or Gln    -   Xaa in position 8 is Gly or Ala    -   Xaa in position 9 is Leu or Ile    -   Xaa in position 10 is Gly, Ser or Ala    -   Xaa in position 11 is Ile or Gly    -   Xaa in position 12 is Ser, Phe or Tyr    -   Xaa_(i) inserted before position 14 is Leu, Ile, Nle    -   Xaa in position 15 is Arg, Lys, Ser or Citrulline (Cit)    -   Xaa in position 19 is Arg, Lys, Ser, Gly or Cit    -   Xaa in position 20 is Gln, Arg or Pro    -   Xaa in position 21 is Ile or leu    -   Xaa in position 22 is Gly, Leu, Ile, Cys or none    -   Xaa in position 23 is Gly or none        wherein the sequence of SEQ ID NO: 4 comprises at least six        consecutive amino acids, -Z- is an optional linker and have the        meaning PEG, modified PEG and/or [Gly]_(n) wherein n=1, 2 or 3,

furthermore two or more of the Cys residues may form part of anintrachain- or interchain disulphide binding, a —S—(CH₂)_(p)—S— or a—(CH₂)_(p)-bridge wherein p=1-8, optionally intervened by one or moreheteroatoms such as O, N or S, Xaa₁ Ile Leu Xaa₄ Xaa₅ Xaa₆ Leu Gly (SEQID NO: 7) Arg Xaa₁₀ Xaa₁₁ -Z- Xaa₁₂ Leu Xaa; Xaa; Xaa₁₄ Xaa₁₅ Xaa₁₆Xaa₁₇ Xaa₁₈ Xaa₁₉ Leu Pro Pro Leuwherein Xaa in position 1 is Phe, Tyr, Trp or Arg

-   -   Xaa in position 4 is Gly, Ser, Asn, Asp, Cys, Val, Thr, Ala, or        Arg    -   Xaa in position 5 is Thr, Ala, Asp, Asn or Ser    -   Xaa in position 6 is Tyr, Cys, Phe, Arg, His, Ser, Val or Leu    -   Xaa in position 10 is Ser, Pro, Phe, Leu or Ile    -   Xaa in position 11 is Ala, Thr, Glu, Gln, Val Pro or Ser    -   Xaa in position 12 is Glu, Lys, Gln, Asp, Asn, Tyr, Trp or Phe    -   Xaa_(i) inserted after position 13 is Ser, Pro, Phe, Leu or Ile    -   Xaa_(i) inserted before position 14 is Ala, Thr, Glu, Gln, Val,        Pro, or Ser    -   Xaa in position 14 is Glu, Lys, Gln, Asp or Asn    -   Xaa in position 15 is Pro, Ser, Ala or Asn    -   Xaa in position 16 is Val, Asn, Gly or Pro    -   Xaa in position 17 is Pro, Gln, His, Ser, Leu, Arg, Thr, Asp or        Ile    -   Xaa in position 18 is Leu Phe or Val    -   Xaa in position 19 is Gln, Leu, Pro, His, Asp or Glu

wherein the sequence of SEQ ID NO: 7 consists of at least sixconsecutive amino acids, the linker -Z- is optional and have the meaningPEG, modified PEG and/or [Gly]_(n) wherein n=1, 2 or 3, Xaa₁ Leu Val GlyXaa₅ Pro Xaa₇ (SEQ ID NO: 10) Xaa₈ Pro Xaa₁₀ Xaa₁₁ Pro -Z-[Arg]_(m) Xaa;Xaa₁₃ Xaa₁₄ Pro Xaa₁₆ Xaa₁₇ Xaa₁₈ Xaa₁₉ Xaa₂₀ Xaa₂₁wherein the Xaa in position 1 is Lys or Arg

-   -   Xaa in position 5 is Phe or Leu    -   Xaa in position 7 is Ile or Val    -   Xaa in position 8 is Thr, Arg, Lys, Ala or Met    -   Xaa in position 10 is Gln or His    -   Xaa in position 11 is Val, Leu or Ile    -   Xaa_(i) inserted before position 13 is Leu    -   Xaa in position 13 is Leu, Val or Thr    -   Xaa in position 14 is Arg or Citrulline (Cit)    -   Xaa in position 16 is Met, Val, Ile or Nle, Leu    -   Xaa in position 17 is Thr or Asp    -   Xaa in position 18 is Tyr, Phe or Arg    -   Xaa in position 19 is Lys or Arg    -   Xaa in position 20 is Ala, Gly, Ser, Glu or Gln    -   Xaa in position 21 is Ala, Ser or Val        wherein the sequence of SEQ ID NO: 10 consists of at least six        consecutive amino acids, the linker -Z- is optional and have the        meaning PEG, modified PEG and/or [Gly]_(n) wherein n=1, 2 or 3        and independently from n, m in [Arg]_(m) is=0, 1, 2 or 3, the        terminal ends of the sequences may be free carboxyl- or amino        groups, amides, acyls, acetyls or salts thereof, and/or the said        peptide sequences are immobilized to a solid support.

The new peptide sequences have the potential to serve as a good antigenwherein the antigen comprises at least one peptide selected from thegroup of sequences of SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7 or SEQ IDNO: 10. The antigenicity may be adapted through adjusting the ratio orconcentration of different peptides or size of the peptides by forinstance dimerization or polymerization and/or immobilization to a solidphase. The antigen comprises one or more polypeptide sequences,according to the invention, which could be either linked by a bridge forinstance a disulphide bridge between the Cys residues of the chains orbridges like C₁-C₈ alkylen possibly intervened by one or moreheteroatoms like O, S, or N or preferably they are unlinked. The chainsmay be immobilized to a solid phase in monomeric, dimeric or oligomericforms. Further amino acids may be added to the ends in order to achievean <<arm>> to facilitate immobilization.

PEG is polyethylene glycol (HO(CH₂CH₂O)_(a) H and can be part of thelinker -Z-, optionally PEG is modified by a dicarboxylic acid(HO(CH₂CH₂O)_(a) CO(CH₂)_(b) COOH) or a terminal carboxylic group(HO(CH₂CH₂O)_(a-1)CH₂COOH) where a=1-10 and b=2-6, prior to linking.

The linker -Z- can either consist of PEG, modified PEG, or a combinationthereof and/or one or more Gly residues combined. Alternatively thelinker -Z- can consist of a Gly-bridge [Gly]_(n) where n=1, 2 or 3.

All amino acids in the peptides of the invention can be in both D- orL-form, although the naturally occurring L-form is preferred.

The C— and N-terminal ends of the peptide sequences could deviate fromthe natural sequences by modification of the terminal NH₂-group and/orCOOH-group, they may for instance be acylated, acetylated, amidated ormodified to provide a binding site for a carrier or another molecule.

The peptides according to the invention are consisting of 6 to 50 aminoacids, preferably between 10 and 30 amino acids. They are covering allnatural variation of amino acids in the identified positions.

The polypeptide antigen according to the invention is either in a freeor in a carrier-bound form. The carrier or solid phase to which thepeptide is optionally bound can be selected from a vide variety of knowncarriers. It should be selected with regard to the intended use of theimmobilized polypeptide as a diagnostic antigen or as an immunizingcomponent in a vaccine.

Examples of carriers that can be used for e.g. diagnostic purposes aremagnetic beads or latex of co-polymers such as styrene-divinyl benzene,hydroxylated styrene-divinyl benzene, polystyrene, carboxylatedpolystyrene, beads of carbon black, non-activated or polystyrene orpolyvinyl chloride activated glass, epoxy-activated porous magneticglass, gelatine or polysaccharide particles or other protein particles,red blood cells, mono- or polyclonal antibodies or fab fragments of suchantibodies.

According to a further embodiment of the present invention, the antigensmay form part of a vaccine possibly combined with carriers, adjuvants orcombined with other immunostimulating elements such as canarypox viruscarrying the env gene. Examples of carriers and/or adjuvants for vaccinepurposes are other proteins such as human or bovine serum albumin andkeyhole limpet haemocyanin and fatty acids. Immunostimulatory materialsmay be divided into three groups; adjuvants, carriers for antigens andvehicles. Examples of adjuvants include aluminum hydroxyd, aluminumsalts, saponin, muramyl di and tripeptides, monophosphoryl lipid A,palmitic acid, B.pertussis and various cytokines including the Th1cytokine IL-12 and IL-1. A number of protein toxins can be used to carrypassenger proteins across cellular membranes into the cytosol, which areuseful in developing CTL vaccines. Carriers include bacterial toxoidssuch as inactivated tetanus and cholera toxins, genetically detoxifiedbacterial toxins such as heat labile enterotoxin from E.coli, fattyacids, live vectors such as polio chimeras and hybrid proteins that formparticulates for example yeast retrotransposon hybrid TY particles andHBcAg particles. Vehicles which are frequently occurring components inmodern vaccines are consisting of mineral oil emulsion, Freunds completeand incomplete adjuvant, vegetable oil emulsions, nonionic blockco-polymer surfactants, squalene or squalane, lipopeptides, liposomesand biodegradable microspheres. Two novel adjuvants which possesssignificant potential for the development of new vaccines include anoil-in-water microemulsion (MF59) and polymeric microparticles. Anysubstance that can enhance the immunogenicity of the antigen may be usedand several further alternatives of carriers or adjuvants are given inthe US or European Pharmacopoeia.

A suitable formulation of the antigen for immunostimulatory uses mayalso comprise interferons such as INF-γ, antiviral chemokines orhaematopoietic growth factors such as granulocyte macrophage growthfactor.

Another approach in order to enhance the stimulation and absorption infor instance the intestine is to administer the peptides of theinvention, with small peptides such as di, tri or tetrapeptides. Thesepeptides can be administered in addition to or in combination with thepeptides of the invention. Preferably the peptides are administeredtogether with the tripeptide YGG, consisting of amino acids in the D- orL-forms, preferably in the D-form.

Recent approaches to non-parenteral delivery of vaccines, for instancevia mucosa include; gene fusion technology to create non-toxicderivatives of mucosal adjuvants, genetically inactivated antigens witha deletion in an essential gene, coexpression of an antigen and aspecific cytokine that is important in the modulation and control of amucosal immune response, and genetic material itself that would allowDNA or RNA uptake and its endogenous expression in the host's cells.

One approach for developing durable responses where cell-mediatedimmunity is required, is to vaccinate with plasmid DNA encoding one ormore specific antigen(s).

In order to protect against HIV infection, vaccines should induce bothmucosal and systemic immune responses and could be administered by anyconvenient route, parenterally or non-parenterally, such assubcutanously, intracutanously, intravenously, intramuscularly,perorally, mucosally or intranasally for example.

In a preferred embodiment of the vaccine according to the presentinvention it comprises antigens containing peptides selected from atleast one of the groups of the SEQ ID NO: 1, 4, 7 and 10, more preferredthe different peptides occur in equal amounts.

In a further preferred embodiment the vaccine composition contains theantigens; F V I H R L E P W L H P G S Q H NI (SEQ ID NO: 14) T A S T N -NH₂ and R L V G F P V K P Q V P G L L R P (SEQ ID NO: 15) L T Y K A A -NH₂.

The sequences can activate the cellular immune system, and contributewith CTL-epitopes. The amino acid changes implemented within the frameof the CTL-epitopes are designed to achieve enhanced binding. Otheramino acid changes have been conducted in order to facilitate thesynthesis of the peptide and/or increase the solubility of the peptide.

A method for detecting antibodies, induced by HIV-1 or HIV-1 specificpeptides or proteins, in a sample of body fluid using the presentantigens is a further embodiment of the invention. Also immunoassay kitdesigned for this detection and antibodies capable of selectivelyreacting with the said antigens are encompassed by the presentinvention.

Description of the Preparation of the Peptides

The peptides of the invention can be produced by any known method ofproducing a linear amino acid sequence, such as recombinant DNAtechniques. A nucleic acid sequence that encodes one or more peptides ofthe invention or a multimer of the said peptides, is introduced into anexpression vector. Suitable expression vectors are for instanceplasmids, cosmids, viruses and YAC (yeast artifical chromosome) whichcomprise necessary control regions for replication and expression. Theexpression vector may be stimulated to expression in a host cell.Suitable host cells are for example bacteria, yeast cells and mammalcells. Such techniques are well known in the art and described forinstance by Sambrook et al., Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1989. Otherwell-known techniques are degradation or synthesis by coupling of oneamino acid residue to the next one in liquid phase or preferably on asolid phase (resin) for instance by the so-called Merrifield synthesis.See for instance Barany and Merrifield in the Peptides, Analysis,Synthesis, Biology, Vol.2, E. Gross and Meinhofer, Ed. (Acad.Press,N.Y., 1980), Kneib-Coronier and Mullen Int. J. Peptide Protein Res., 30,p.705-739 (1987) and Fields and Noble Int.J.Peptide Protein Res., 35,p.161-214 (1990).

In case a linked or cyclic peptide is desired, the amino acid sequenceis subjected to a chemical oxidation step in order to cyclize or linkthe two cysteine residues within one or between two peptide sequences,when the appropriate linear amino acid sequences are synthesized, seeAkaji et al., Tetrahedron Letter, 33, 8, p.1073-1076, 1992.

General Description of Synthesis

All peptide derivatives prepared in the Examples given below weresynthesized on a Milligen 9050 Peptide Synthesizer using a standardprogram. The resin used was Tenta Gel P RAM with a theoretical loadingof 0.20 meq/g (RAPP POLYMERE GmbH, Tübingen). The final product of thesynthesis was dried in vacuuo overnight. The peptide was then cleavedfrom the resin by treatment with 90% trifluoroacetic acid in thepresence of ethandithiol (5%) and water (5%) as scavengers (1.5 hours atRT). Then the resin was filtered and washed on filter with additionaltrifluoro acetic acid (100%) (2×20 ml). The combined filtrates wereevaporated in vacuuo (water bath at RT) and the residue was trituratedwith ethyl ether (200 ml) and the precipitated product filtered off. Thesolid was promptly dissolved on filter with glacial acetic acid (100 ml)and added to 1.5 l of 20% acetic acid in methanol and treated with 0.1 Msolution of iodine in methanol until a faint brown colour remained. ThenDowex 1×8 ion exchange in acetate form (15 g) (Bio-Rad, Richmond,Calif.) was added and the mixture filtered. The filtrate was evaporatedand the residue freeze-dried from acetic acid. The product was thenpurified by reversed phase liquid chromatography on a column filled withKromasil® 100-5 C8 (EKA Nobel, Surte, Sweden) in a suitable systemcontaining acetonitrile in 0.1% trifluoro acetic acid water solution.The samples collected from the column were analyzed by analytical highperformance liquid chromatography (HPLC) (Beckman System Gold, U.S.A.)equipped with a Kromasil® 100-5 C8 Column (EKA Nobel, Surte, Sweden).Fractions containing pure substance were pooled, the solvent wasevaporated and the product freeze-dried from acetic acid. The final HPLCanalysis was performed on final product, and the structure of thepeptide was confirmed by amino acid analysis and mass spectrometry(LDI-MS).

All amino acids used during the synthesis were L-amino acids and theywere protected with a fluorenyl methoxy-carbonyl group at the α-aminofunction. The side chains were protected as follows:Cys (Trt), Gln(Trt), Glu(OtBu), Thr(tBu).

The abbreviations, within the brackets are:

-   -   Trt=triphenyl methyl    -   t-Bu=tert. Butyl    -   OtBu=tert. Butyl ester

The amino acid derivatives were supplied by Bachem AG, Switzerland.

EXAMPLE 1

Preparation of C S W V N P R L E P W NI H P G S Q H NI T A C T N—NH₂(SEQ ID NO: 2). The peptide was synthesized in amide form, from thecorresponding starting materials according to the general description ofsynthesis. The peptide was then cyclizated by oxidation with I₂. Thepeptide was dissolved in acetic acid/methanol (1:4) and 0.1 M I₂ inmethanol was added. The purity was determined by HPLC analysis and thestructure was confirmed by amino acid analysis and mass spectrometry(LDI-MS).

Purity (HPLC): more than 97% (single impurity less than 1%) Molecularweight (free base): 2746.2

EXAMPLE 2

Preparation of F V I P R L E P W NI H P G S Q P NI T A C T N—NH₂(SEQ IDNO: 3). The peptide was synthesized in amide form, from thecorresponding starting materials according to the general description ofsynthesis. The purity was determined by HPLC analysis and the structurewas confirmed by amino acid analysis and mass spectrometry (LDI-MS).

Purity (HPLC): more than 97% (single impurity less than 1%)

Molecular weight (free base): 2538.0

EXAMPLE 3

Preparation o f Y L L F L T K G L G I S G G G Y NI G Cit K K R Cit Q I LG-NH₂ (SEQ ID NO: 5). The peptide is synthesized in amide form, from thecorresponding starting materials according to the general description ofsynthesis. The purity is determined by HPLC analysis and the structureis confirmed by amino acid analysis and mass spectrometry (LDI-MS).

EXAMPLE 4

Preparation of Y L NI F L T R G L G I S G G G Y NI G Cit K K R Cit Q ICG-NH₂ (SEQ ID NO: 6). The peptide was synthesized in amide form, fromthe corresponding starting materials according to the generaldescription of synthesis. The purity was determined by HPLC analysis andthe structure was confirmed by amino acid analysis and mass spectrometry(LDI-MS).

Purity (HPLC): more than 97% (single impurity less than 1%) Molecularweight (free base): 3097.7

EXAMPLE 5

Preparation of R I L S T Y L G R I S G G G W L S A E P V P L Q L P PL-NH₂ (SEQ ID NO: 8). The peptide was synthesized in amide form, fromthe corresponding starting materials according to the generaldescription of synthesis. The purity was determined by HPLC analysis andthe structure was confirmed by amino acid analysis and mass spectrometry(LDI-MS).

Purity (HPLC): more than 97% (single impurity less than 1%) Molecularweight (free base): 2990.6

EXAMPLE 6

Preparation of R I L S T Y L G R I S G G G Y L S A E P V P L Q L P PL-NH₂ (SEQ ID NO: 9). The peptide is synthesized in amide form, from thecorresponding starting materials according to the general description ofsynthesis. The purity is determined by HPLC analysis and the structureis confirmed by amino acid analysis and mass spectrometry (LDI-MS).

EXAMPLE 7

Preparation of K L V G F P V K P Q V P G G G R L L Cit P NI T Y K AA-NH₂ (SEQ ID NO: 11). The peptide is synthesized in amide form, fromthe corresponding starting materials according to the generaldescription of synthesis. The purity is determined by HPLC analysis andthe structure is confirmed by amino acid analysis and mass spectrometry(LDI-MS).

EXAMPLE 8

Preparation of R L V G F P V K P Q V P G G G R L L R P L T Y K A A-NH₂(SEQ ID NO: 12). The peptide was synthesized in amide form, from thecorresponding starting materials according to the general description ofsynthesis. The purity was determined by HPLC analysis and the structurewas confirmed by amino acid analysis and mass spectrometry (LDI-MS).

Purity (HPLC): more than 97% (single impurity less than 1%) Molecularweight (free base): 2790.4 Molecular formula: C₁₃₀H₂₁₇O₃₉N₂₉

EXAMPLE 9

Dimerisation Via Disulphide Bridge.

The peptide sequences of the Example 2 are linked via an oxidation stepto form a dipeptide wherein the cysteine residues form a disulphidebridge. The bridge is formed in either ways;

-   -   A) Oxidation with I₂. The peptides (equal amounts if different)        are dissolved in acetic acid/methanol (1:4) and 0.1 M I₂ in        methanol is added yielding a mixture of the homodimer, or    -   B) Oxidation via [Cys(Spy)²²]-SEQ ID NO: 3. 2.3 mM of the        peptide of SEQ ID NO: 3 dissolved in 2 M AcOH (aq) and        2-propanol (1:1) is treated with 2,2 dithiodipyridin (3 eqv) to        yield [Cys(Spy)²²]-SEQ ID NO: 3. [Cys(Spy)²²]-SEQ ID NO: 3 is        dissolved in 10 mM NH₄Oac (aq pH=6, 5) and methanol (5:2) to        yield the dimer of SEQ ID NO: 13.

The purity of the resulting dimer is determined by HPLC analysis and thestructure is confirmed by amino acid analysis and mass spectrometry(LDI-MS).

EXAMPLE 10

Preparation of F V I H R L E P W L H P G S Q H NI T A S T N—NH₂(SEQ IDNO:14). The peptide was synthesized in amide form, from thecorresponding starting materials according to the general description ofsynthesis. The purity was determined by HPLC analysis and the structurewas confirmed by amino acid analysis and mass spectrometry (LDI-MS).

Purity (HPLC): more than 98% (single impurity less than 1%) Molecularweight (free base): 2540.2

EXAMPLE 11

Preparation of R L V G F P V K P Q V P G L L R P L T Y K A A-NH₂ (SEQ IDNO:15). The peptide was synthesized in amide form, from thecorresponding starting materials according to the general description ofsynthesis. The purity was determined by HPLC analysis and the structurewas confirmed by amino acid analysis and mass spectrometry (LDI-MS).

Purity (HPLC): more than 99% (single impurity less than 1%) Molecularweight (free base): 2520.3

EXAMPLE 12 REFERENCE EXAMPLE

Preparation of a nativ tat1 sequence; M E S V D P R L E P W K H P G S QP K T A C T N—NH₂ (SEQ ID NO:16). The peptide was synthesized in amideform, from the corresponding starting materials according to the generaldescription of synthesis. The purity was determined by HPLC analysis andthe structure was confirmed by amino acid analysis and mass spectrometry(LDI-MS).

Purity (HPLC): more than 97% (sing le impurity less than 1%) Molecularweight (free base): 2708.1

EXAMPLE 13 REFERENCE EXAMPLE

Preparation of a nativ tat2 sequence; Q V C F I T K G L G I S Y G R K KR R Q R R R—NH₂ (SEQ ID NO:17). The peptide was synthesized in amideform, from the corresponding starting materials according to the generaldescription of synthesis. The purity was determined by HPLC analysis andthe structure was confirmed by amino acid analysis and mass spectrometry(LDI-MS).

Purity (HPLC): more than 94% Molecular weight (free base): 2806.4

EXAMPLE 14 REFERENCE EXAMPLE

Preparation of a nativ Nef sequence; E E V G F P V R P Q V P L R P M T YK A A-NH₂ (SEQ ID NO:18). The peptide was synthesized in amide form,from the corresponding starting materials according to the generaldescription of synthesis. The purity was determined by HPLC analysis andthe structure was confirmed by amino acid analysis and mass spectrometry(LDI-MS).

Purity (HPLC): more than 95% Molecular weight (free base): 2384.8

EXAMPLE 15

A vaccine comprising the peptides of the SEQ ID NO: 3 and 12 isprepared. The freeze-dried peptides are dissolved in sterile water at afinal concentration of 4 mg/ml. A preparation of agranulocyte-macrophage-colony stimulating factor (GM-CSF) is alsoprepared, according to the manufacturers directions for use, to a finalconcentration of 0.3 mg/ml. The two solutions are administeredintracutaneously. A typical injection dose is 100 μl.

EXAMPLE 16

A vaccine comprising the peptides of the SEQ ID NOS: 14 and 15 isprepared. The freeze dried peptides are dissolved in sterile water at afinal concentration of 4 mg/ml. A preparation of agranulocyte-macrophage-colony stimulating factor (GM-CSF) is alsoprepared, according to the manufacturers directions for use, to a finalconcentration of 0.3 mg/ml. The two solutions are administeredintracutaneously. A typical injection dose is 100 μl.

EXAMPLE 17

An antigen solution or suspension is mixed with equal parts of Freund'sadjuvant of Behring, complete or incomplete, and is then finelyemulsified by being drawn up into, and vigorously pressed out of, aninjection syringe, or with a homogenator. The emulsion should remainstable for at least 30 minutes. The antigen-adjuvant emulsions is bestinjected subcutaneously as a depot.

EXAMPLE 18

Immunoassay for Detection of Antibodies Induced by HIV-1.

The magnetic particle reagents are to be prepared according to themanufacturers recommended protocol. Dynal AS, is the manufacturer of theDynabeads, which are employed. The magnetic particles coated with ligandare called Reagent 1. A peptide according to the invention is covalentlycoupled to the pre-activated surface of the magnetic particles. It isalso possible to physically absorb the peptide to the surface of themagnetic particles. The concentration of particles in Reagent 1 iswithin the range from 1 mg/ml to 15 mg/ml. The particle size variesbetween 0.2 μm to 15 μm. The concentration of peptides is within therange from 0.01 mg/mg particle to 1 mg/mg particle.

The anti human Ig Alkaline Phosphatase (AP) conjugated antibody reagentis prepared according to the recommended protocol of Dako AS. Thisprotocol is a standard procedure in this field. This reagent is calledReagent 2.

The substrate solution phenolphtaleine-monophosphate is to be preparedaccording to the recommended protocol of Fluka AG. This protocol is astandard procedure in this field. The substrate solution is calledReagent 3.

The washing and incubation buffer which is used is standard 0,05Mtris-base buffer with the following additional compounds; Tween 20(0.01% to 0.1%), glycerol (0.1% to 10%) and sodium chloride (0.2% to0.1%).

The assay procedure comprises an incubation step wherein 1 drop ofReagent 1 is mixed with 2 drops of washing buffer in each well. Aftermixing, 30 μl of sample is added and the solution is incubated for 5minutes. The magnetic particles can be trapped by a magnet and theliquid removed, before the magnet is separated. Then the wells arewashed twice in 4 drops of washing solution, before incubation withReagent 2. 1 drop of Reagent 2 is added with 2 drops of washing bufferand the solution is incubated for 5 minutes. The magnetic particles canbe trapped by a magnet and the liquid removed, before the magnet isseparated. Then the washing step is repeated before incubation withReagent 3. 2 drops of Reagent 3 is added to each well and the solutionis incubated for 3 minutes. The results can be read against a whitebackground. Positive results are red (3+=strong red) whereas negativeresults are clearly light yellow/brown solutions as obtained in thenegative control.

The immunoassay kit could be used in detection of antibodies, inducedeither by HIV virus or HIV-specific peptides or proteins, for instancethe peptides of the present invention.

EXAMPLE 19

Therapeutic or Prophylactic Vaccine.

At least one of the polypeptides of the invention, selected from thegroup of sequences SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7 or SEQ IDNO: 10 can form antigens and constitute the active principle of aprophylactic or therapeutic vaccine intended to provide protectionagainst the human immunodeficiency virus type 1 (HIV-1). The vaccine mayinclude compounds having beneficial effects in protecting or stimulatingthe hosts immune system (human being or vertebrate animal) for instanceinterleukins, interferons, granulocyte macrophage growth factors,haematopoietic growth factors or similar. Preferably the vaccinecomposition further contain an adjuvant or vehicle, more preferably theadjuvant or vehicle is Monophosphoryl Lipid A (MPL®) possibly with alum,Freund's adjuvant (complete or incomplete) or aluminum hydroxyd. Theoptimal amount of adjuvant/vehicle will depend on the type(s) which ischosen.

The peptides of the invention might be modified by C-terminal additionof a single fatty acid such as a single paimitoyl chain to form alipopeptide vaccine. Further the lipopeptides can be introduced intoliposome membranes by the freeze-thaw method resulting in liposomesbearing the peptide ligands on their surface.

The peptide or vaccine formulation can be freeze-dried prior to storage.The freeze-dried peptides can be dissolved in sterile water to a finalconcentration of 0.1-100 mg/ml. The vaccine may be stored preferably atlow temperature, in ampoules containing one or more dosage units, readyfor use. A typical dosage unit of the peptide according to the inventionis within the concentration range: 0.05 μg-1 mg per kg bodyweight,preferably within 0, 15 μg-0.15 mg per kg body weight. Persons skilledin the art will appreciate that a suitable dose will depend on the bodyweight of the pasient, the type of disease, severity of condition,administration route and several other factors. When used as atherapeutic vaccine, the vaccine might be administered up to 12 times,through injections. Further boosters might follow and can in some casestake place throughout the patients life. In preparation of an injectionsolution the peptides are dissolved in sterile water at a finalconcentration of 1 mg/ml per peptide. Typically an injection volume is100 μl to 200 μl (2×100 μl). The peptide is preferably co-administeredwith a suitable adjuvant and/or a granulocyte-macrophage growth factorfor instance Leucomax® <<Shering Plough>> made within a concentrationrange of from 0.1 mg/ml to 1 mg/ml, or according to the manufacturersrecommendations for use. Particularly preferred is a combination therapywhere the present peptides are administered together with the peptidesas described in the published International patent application no.PCT/NO00/00075 and/or the co-pending Norwegian patent application no.2000 4413. These peptides may be administered simultaneously orsequentially. Suitable administration may be intracutane, subcutane,intravenous, peroral, intramuscular, intranasal, mucosal or any othersuitable route. Booster administrations may be required in order tomaintain protection. For persons skilled in the art it will beunderstood that the vaccine compositions according to the invention areuseful not only in prevention of infection, but also in treatment ofinfection.

No toxic effects of the peptides according to the invention, areobserved when injected in mice in a dosage of 100 μg per kg body weight.

The above Examples are only meant as illustrating the invention. It mustbe understood that a person skilled in the art can modify the peptides,antigens and vaccines herein described without deviating from theconcept and scope of this invention as set forth in the claims.

1. Peptide derived from HIV-1 regulatory and auxiliary protein, characterized in that it comprises at least one amino acid sequence which contains modifications compared with the native sequence and is selected from the groups of amino acid sequences: Xaa₁ Xaa₂ Xaa₃ Xaa₄ Xaa₅ Xaa₆ Xaa₇ (SEQ ID NO: 1) Leu Glu Pro Trp Xaa₁₂ His Pro Xaa₁₅ Xaa₁₆ Xaa₁₇ Xaa₁₈ Xaa₁₉ Xaa₂₀ Xaa₂₁ Xaa₂₂ Xaa₂₃ Xaa₂₄

wherein the amino acids of the chain have the following meanings; Xaa in position 1 of the peptide derivative is Met, Ser, Cys or none, Xaa in position 2 is Glu, Asp, Val, Ser or none Xaa in position 3 is Trp, Tyr or Phe, Xaa in position 4 is Val or Ile, Xaa in position 5 is Ile, Xaa in position 6 is Pro, His or Ala, Xaa in position 7 is Arg, Asn, Ser, Lys, Glu or Asp Xaa in position 12 is Leu, Ile or Nle Xaa in position 15 is Gly or Pro Xaa in position 16 is Ser, Asn or Ala, Xaa in position 17 is Gln, Lys or Thr Xaa in position 18 is Pro or His, Xaa in Position 19 is Leu, Ile or Nle Xaa in position 20 is Thr, Ala or Ile Xaa in position 21 is Ala, Pro, Asp or Val Xaa in position 22 is Cys or Ser Xaa in position 23 is Thr, Asn or Ser Xaa in position 24 is Asn, Lys, Arg, Gln, Ala Pro or Thr the peptide comprises at least six consecutive amino acids of the sequence of SEQ ID NO: 1, Xaa₁ Xaa₂ Xaa₃ Phe Xaa₅ Xaa₆ Xaa₇ (SEQ ID NO: 4) Xaa₈ Xaa₉ Xaa₁₀ Xaa₁₁ Xaa₁₂ -Z- Tyr Xaa; Gly Xaa₁₅ Lys Lys Arg Xaa₁₉ Xaa₂₀ Xaa₂₁ Xaa₂₂ Xaa₂₃

wherein the amino acids of the chain have the following meaning; Xaa in position 1 is Pro, Ile, Leu, Thr, Tyr or Val Xaa in position 2 is Val, Ala Cys, Leu, Xaa in position 3 is Cys, Ile, Leu, Val or Nle Xaa in position 5 is Ile, Leu, Gln, Met or Thr Xaa in position 6 is Thr, Asn, Lys or Arg Xaa in position 7 is Lys, Arg or Gln Xaa in position 8 is Gly or Ala Xaa in position 9 is Leu or Ile Xaa in position 10 is Gly, Ser or Ala Xaa in position 11 is Ile or Gly Xaa in position 12 is Ser, Phe or Tyr Xaa_(i) inserted before position 14 is Leu, Ile, Nle Xaa in position 15 is Arg, Lys, Ser or Citrulline (Cit) Xaa in position 19 is Arg, Lys, Ser, Gly or Cit Xaa in position 20 is Gln, Arg or Pro Xaa in position 21 is Ile or leu Xaa in position 22 is Gly, Leu, Ile, Cys or none Xaa in position 23 is Gly or none wherein the sequence of SEQ ID NO: 4 comprises at least six consecutive amino acids, —Z— is an optional linker and have the meaning PEG, modified PEG and/or-[Gly]_(n) wherein n=1, 2 or 3, Xaa₁ Ile Leu Xaa₄ Xaa₅ Xaa₆ Leu Gly (SEQ ID NO: 7) Arg Xaa₁₀ Xaa₁₁ -Z- Xaa₁₂ Leu₁₃ Xaa; Xaa; Xaa₁₄ Xaa₁₅ Xaa₁₆ Xaa₁₇ Xaa₁₈ Xaa₁₉ Leu Pro Pro Leu

wherein Xaa in position 1 is Phe, Tyr, Trp or Arg Xaa in position 4 is Gly, Ser, Asn, Asp, Cys, Val, Thr, Ala, or Arg Xaa in position 5 is Thr, Ala, Asp, Asn or Ser Xaa in position 6 is Tyr, Cys, Phe, Arg, His, Ser, Val or Leu Xaa in position 10 is Ser, Pro, Phe, Leu or Ile Xaa in position 11 is Ala, Thr, Glu, Gln, Val, Pro or Ser Xaa in position 12 is Glu, Lys, Gln, Asp, Asn, Tyr, Trp or Phe Xaa_(i) inserted after position 13 is Ser, Pro, Phe, Leu or Ile Xaa_(i) inserted before position 14 is Ala, Thr, Glu, Gln, Val, Pro, or Ser Xaa in position 14 is Glu, Lys, Gln, Asp or Asn Xaa in position 15 is Pro, Ser, Ala or Asn Xaa in position 16 is Val, Asn, Gly or Pro Xaa in position 17 is Pro, Gln, His, Ser, Leu, Arg, Thr, Asp or Ile Xaa in position 18 is Leu Phe or Val Xaa in position 19 is Gln, Leu, Pro, His, Asp or Glu wherein the sequence of SEQ ID NO: 7 consists of at least six consecutive amino acids, the linker -Z- is optional and have the meaning PEG, modified PEG and/or [Gly]_(n) wherein n=1,2 or 3, (SEQ ID NO: 10) Xaa₁ Leu Val Gly Xaa₅ Pro Xaa₇ Xaa₈ Pro Xaa₁₀ Xaa₁₁ Pro -Z-[Arg]_(m) Xaa_(i) Xaa₁₃ Xaa₁₄ Pro Xaa₁₆ Xaa₁₇ Xaa₁₈ Xaa₁₉ Xaa₂₀ Xaa₂₁

wherein the Xaa in position 1 is Lys or Arg Xaa in position 5 is Phe or Leu Xaa in position 7 is Ile or Val Xaa in position 8 is Thr, Arg, Lys, Ala or Met Xaa in position 10 is Gln or His Xaa in position 11 is Val, Leu or Ile Xaa_(l) inserted before position 13 is Leu Xaa in position 13 is Leu, Val or Thr Xaa in position 14 is Arg or Citrulline (Cit) Xaa in position 16 is Met, Val, Ile or Nle, Leu Xaa in position 17 is Thr or Asp Xaa in position 18 is Tyr, Phe or Arg Xaa in position 19 is Lys or Arg Xaa in position 20 is Ala, Gly, Ser, Glu or Gln Xaa in position 21 is Ala, Ser or Val wherein the sequence of SEQ ID NO: 10 consists of at least six consecutive amino acids, the linker -Z- is optional and have the meaning PEG, modified PEG and/or [Gly]_(n) wherein n=1, 2 or 3 and independently from n, m in [Arg]_(m) is 0, 1, 2 or 3, the terminal ends of the sequences may be free carboxyl- or amino groups, amides, acyls, acetyls or salts thereof, two or more of the Cys residues may form part of an intrachain- or interchain disulphide binding, a —S—(CH₂)_(p)—S— or a —(CH₂)_(p)-bridge wherein p=1-8 optionally intervened by one or more heteroatoms such as O, N and S and/or the said peptide sequences are immobilized to a solid support.
 2. Peptide according to claim 1, characterized in that the amino acid sequence of SEQ ID NO: 1 is selected from the groups of SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO:
 14. 3. Peptide according to claim 1, characterized in that the amino acid sequence of SEQ ID NO: 4 is selected from the groups of SEQ ID NO: 5and SEQ ID NO:6.
 4. Peptide according to claim 1, characterized in that the amino acid sequence of SEQ ID NO: 7 is selected from the groups of SEQ ID NO: 8 and SEQ ID NO:
 9. 5. Peptide according to claim 1, characterized in that the amino acid sequence of SEQ ID NO: 10 is selected from the groups of SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO:
 15. 6. Antigen, characterized in that it comprises at least one peptide according to claim
 1. 7. Antigen according to claim 6, characterized in that it comprises at least one peptide selected from at least one of the groups SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7 and SEQ ID NO:
 10. 8. Vaccine composition, characterized in that it comprises an antigen according to claim 6 with a pharmaceutically acceptable diluent and optionally an adjuvant, carrier and/or vehicle and optionally additional immunostimulatory compound(s).
 9. Vaccine composition according to claim 8, characterized in that it comprises at least one peptide selected from at least one of the groups of SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7 and SEQ ID NO:
 10. 10. Vaccine composition according to claim 9, characterized in that it comprises the peptides of the SEQ ID NO: 14 and SEQ ID NO:
 15. 11. Vaccine composition according to the claims 8-10, characterized in that the peptides are dissolved in a sterile water solution and the optional immunostimulatory compound is a granulocyte macrophage colony stimulating factor.
 12. Vaccine composition according to the claims 8-11, characterized in that the composition comprises an adjuvant selected from the group Monophosphoryl Lipid A (MPL®), Freund's complete or incomplete adjuvant or aluminum hydroxyd.
 13. Vaccine composition, characterized in that an antigen according to claim 6 is formulated as a lipopeptide and/or a liposome formulation.
 14. A method of detecting antibodies, induced by a HIV or HIV-specific peptides or proteins, in a sample of body fluid, characterized in that subjecting the said sample to an immunoassay, wherein the antigen(s) is/are selected from the peptides of the claims 1, 2, 3, 4 and
 5. 15. An immunoassay kit for the detection of antibodies, induced by a HIV or HIV-specific peptides or proteins, in a sample of body fluid, characterized in that the diagnostic antigen is a peptide of any one of the previous claims 1 to
 5. 16. Antibody, characterized in that it is capable of selectively reacting with the antigen of the claims 6 and
 7. 